Summary of Study ST003247

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002016. The data can be accessed directly via it's Project DOI: 10.21228/M81C00 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003247
Study TitleEffects of mitoregulin loss in aged female mice
Study SummaryCardiac lipidome analysis in aged (21 to 23-months old) female wildtype and mtln knockout mice
Institute
University of Iowa
Last NameBoudreau
First NameRyan
Address4334 PBDB, 169 Newton Rd, Iowa City, IA 52242
Emailryan-boudreau@uiowa.edu
Phone3193535573
Submit Date2024-06-04
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-12-31
Release Version1
Ryan Boudreau Ryan Boudreau
https://dx.doi.org/10.21228/M81C00
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002016
Project DOI:doi: 10.21228/M81C00
Project Title:Effects of mitoregulin loss on cardiac and mitochondrial lipids in mice
Project Summary:We and others discovered a highly-conserved mitochondrial transmembrane microprotein, named Mitoregulin (Mtln), that supports lipid metabolism. We reported that Mtln strongly binds cardiolipin (CL), increases mitochondrial respiration and Ca2+ retention capacities, and reduces reactive oxygen species (ROS). Here we extend our observation of Mtln-CL binding and examine Mtln influence on cristae structure and mitochondrial membrane integrity during stress. We demonstrate that mitochondria from constitutive- and inducible Mtln-knockout (KO) mice are susceptible to membrane freeze-damage and that this can be rescued by acute Mtln re-expression. In mitochondrial-simulated lipid monolayers, we show that synthetic Mtln decreases lipid packing and monolayer elasticity. Lipidomics revealed that Mtln-KO heart tissues show broad decreases in 22:6-containing lipids and increased cardiolipin damage/remodeling. Lastly, we demonstrate that Mtln-KO mice suffer worse myocardial ischemia-reperfusion injury, hinting at a translationally-relevant role for Mtln in cardioprotection. Our work supports a model in which Mtln binds cardiolipin and stabilizes mitochondrial membranes to broadly influence diverse mitochondrial functions, including lipid metabolism, while also protecting against stress.
Institute:University of Iowa
Last Name:Boudreau
First Name:Ryan
Address:4334 PBDB, 169 Newton Rd, Iowa City, IA 52242
Email:ryan-boudreau@uiowa.edu
Phone:3193535573

Subject:

Subject ID:SU003366
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Gender:Female
Species Group:Mammals

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Sample source
SA3534631133-1KO heart
SA3534641143-2KO heart
SA3534651143-3KO heart
SA3534661145-2KO heart
SA3534671152-1KO heart
SA3534681066-23WT heart
SA3534691066-4WT heart
SA3534701074-34WT heart
SA3534711103-12WT heart
SA353472WTFWT heart
Showing results 1 to 10 of 10

Collection:

Collection ID:CO003359
Collection Summary:Mtln-KO and WT mice were fasted for 4-6 h, and while under deep ketamine/xylazine anesthesia, the heart apex was snipped off and immediately snap-frozen by LN2 immersion.
Sample Type:Heart

Treatment:

Treatment ID:TR003375
Treatment Summary:No treatment.

Sample Preparation:

Sampleprep ID:SP003373
Sampleprep Summary:Lipid extraction. Lipid extraction, based on Matyash et al.72, was performed as follows. All solutions were pre-chilled on ice. Tissues or mitochondrial pellets were transferred to labeled bead-mill tubes (1.4 mm, MoBio Cat# 13113-50) where lipids were extracted in a solution of 250 µL PBS, 225 µL MeOH containing internal standards, and 750 µL MTBE (methyl tert-butyl ether). Internal standards were Avanti SPLASH LipidoMix (Lot#12) at 10 µL per sample and Cambridge Isotope laboratories NSK-B and NSK-B-G1 (deuterated carnitines) at 10 µL per sample. The samples were homogenized in one 30 s cycle using the Omni Bead Ruptor followed by a rest on ice for 1 h. An addition of 188 µL PBS was made to induce phase separation. After centrifugation at 16,000 g for 5 minutes at 4 °C, the upper phases were collected and evaporated to dryness under a gentle nitrogen stream at room temperature. Lipid samples were reconstituted in 500 µL IPA (isopropyl alcohol) and transferred to an LC-MS vial with insert (Agilent 5182-0554 and 5183-2086) for analysis. Concurrently, a process blank sample and pooled quality control (QC) sample was prepared by taking equal volumes (~50 µL) from each sample after final resuspension.

Combined analysis:

Analysis ID AN005318 AN005319
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Agilent 1290 Infinity Agilent 1290 Infinity
Column Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Agilent 6530 QTOF Agilent 6530 QTOF
Ion Mode POSITIVE NEGATIVE
Units pmol lipid per mg tissue pmol lipid per mg tissue

Chromatography:

Chromatography ID:CH004022
Chromatography Summary:Positive Mode RP LCMS
Instrument Name:Agilent 1290 Infinity
Column Name:Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:65
Flow Gradient:Started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min.
Flow Rate:0.4 mL min
Solvent A:40% water/60% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:90% isopropanol/9% acetonitrile/1% water; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:Reversed phase
  
Chromatography ID:CH004023
Chromatography Summary:Negative Mode RP LCMS
Instrument Name:Agilent 1290 Infinity
Column Name:Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:65
Flow Gradient:Started at 15% mobile phase B then increased to 30% B over 2.4 min. It sequentially increased to 48% B from 2.4 – 3.0 min, 82% B from 3 – 13.2 min, and 99% B from 13.2 – 13.8 min where it’s held until 16.7 min and returned to the initial conditions and equilibrated for 5 min.
Flow Rate:0.4 mL min
Solvent A:40% water/60% acetonitrile; 10 mM ammonium formate
Solvent B:90% isopropanol/9% acetonitrile/1% water; 10 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS005048
Analysis ID:AN005318
Instrument Name:Agilent 6530 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:For positive mode, the source gas temperature was set to 225 °C, with a drying gas flow of 11 L/minute, nebulizer pressure of 40 psig, sheath gas temp of 350 °C and sheath gas flow of 11 L/minute. VCap voltage is set at 3500 V, nozzle voltage 500V, fragmentor at 110 V, skimmer at 85 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis.
Ion Mode:POSITIVE
  
MS ID:MS005049
Analysis ID:AN005319
Instrument Name:Agilent 6530 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:For negative mode, the source gas temperature was set to 300 °C, with a drying gas flow of 11 L/minute, a nebulizer pressure of 30 psig, sheath gas temp of 350 °C and sheath gas flow 11 L/minute. VCap voltage was set at 3500 V, nozzle voltage 75 V, fragmentor at 175 V, skimmer at 75 V and octopole RF peak at 750 V. For data processing, Agilent MassHunter (MH) Workstation and software packages MH Qualitiative and MH Quantitative were used. The pooled QC (n=8) and process blank (n=4) were injected throughout the sample queue to ensure the reliability of acquired lipidomics data. For lipid annotation, accurate mass and MS/MS matching was used with the Agilent Lipid Annotator library. Results from the positive and negative ionization modes from Lipid Annotator were merged based on the class of lipid identified. Data exported from MH Quantitative was evaluated using Excel where initial lipid targets are parsed based on the following criteria. Only lipids with relative standard deviations (RSD) less than 30% in QC samples are used for data analysis. Additionally, only lipids with background AUC counts in process blanks that are less than 30% of QC are used for data analysis. The parsed excel data tables are normalized based on the ratio to class-specific internal standards, then to sum prior to statistical analysis.
Ion Mode:NEGATIVE
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